Tau seed amplification assay reveals relationship between seeding and pathological forms of tau in Alzheimer's disease brain.

Tau seed amplification assays (SAAs) directly measure the seeding activity of tau and would therefore be ideal biomarkers for clinical trials targeting seeding-competent tau in Alzheimer's disease (AD). However, the precise relationship between tau seeding measured by SAA and the levels of pathological forms of tau in the AD brain remains unknown. We developed a new tau SAA based on full-length 0N3R tau with sensitivity in the low fg/ml range and used it to characterize 103 brain samples from three independent cohorts. Tau seeding clearly discriminated between AD and control brain samples. Interestingly, seeding was absent in Progressive Supranuclear Palsy (PSP) putamen, suggesting that our tau SAA did not amplify 4R tau aggregates from PSP brain. The specificity of our tau SAA for AD brain was further supported by analysis of matched hippocampus and cerebellum samples. While seeding was detected in hippocampus from Braak stages I-II, no seeding was present in AD cerebellum that is devoid of tau inclusions. Analysis of 40 middle frontal gyrus samples encompassing all Braak stages showed that tau SAA seeding activity gradually increased with Braak stage. This relationship between seeding activity and the presence of tau inclusions in AD brain was further supported by robust correlations between tau SAA results and the levels of phosphorylated tau212/214, phosphorylated tau181, aggregated tau, and sarkosyl-insoluble tau. Strikingly, we detected tau seeding in the middle frontal gyrus already at Braak stage II-III, suggesting that tau SAA can detect tau pathology earlier than conventional immunohistochemical staining. In conclusion, our data suggest a quantitative relationship between tau seeding activity and pathological forms of tau in the human brain and provides an important basis for further development of tau SAA for accessible human samples.

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get.

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to α-syn (α-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-α-syn); (2) Vn-α-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-α-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of α-syn-Vc in Vn-α-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the α-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.